A method for the high spatial resolution imaging of a structure of interest in a specimen with the steps specified above, which is referred to as RESOLFT (REversible Saturable OpticaL Fluorescence Transition), is known from US 2004/0212799 A1 and US 2006/0038993 A1. Here, when converting the substance into the second state by the switching signal, only a defined spatial region of the specimen is respectively omitted deliberately. This region is an intensity minimum of an interference pattern with a zero position, and the intensity of the switching signal is already so large everywhere in the vicinity of the zero position that it exceeds a saturation threshold for complete switching of the substance into the second state. In this way an optical measurement signal, which comes from the fraction of the substance remaining in the first state, can be assigned to the specimen's region deliberately omitted by the switching signal. The spatial resolution for imaging the specimen's structure of interest, which is marked with the substance, therefore no longer depends on the spatial resolution limit of the imaging of the specimen onto the sensor array being used. Rather the spatial resolution is defined by the extent of the zero position of the switching signal, within which the substance still lies in the first state, since there is no measurement signal which can come from the vicinity of the zero position and accordingly needs to be assigned to a spatial position separable from the position of zero position. When spatially imaging the structure of interest in a specimen, it is therefore possible to go below the resolution limit (i.e. the Abbe limit due to diffraction, which is given by the wavelength of the light divided by two times the numerical aperture) which in principle restricts the spatial resolution of the imaging optical methods and depends directly on the wavelength of the longest-wave relevant optical signal.
The substances used in the above-described RESOLFT method for marking the structure of interest in the specimen are switchable fluorescent dyes. This is explained in US 2004/0212799 A1 and US 2006/0038993 A1 in that they are selected from a group of substances which can be converted repeatedly by a switching signal from a first state having first optical properties into a second state having second optical properties, and which can return from the second state into the first state, the two states differing at least in respect of one of the following criteria: conformational state of a molecule; structural formula of a molecule; spatial arrangement of atoms within a molecule; spatial arrangement of bonds within a molecule; accumulation of further atoms or molecules on a molecule; grouping of atoms and/or molecules; spatial orientation of a molecule; mutual orientation of neighboring molecules and ordering formed by a multiplicity of molecules and/or atoms.
The placement of the zero position of the switching signal within the specimen can be determined from the intensity distribution of the measurement signal over the sensor array with an accuracy higher than the spatial resolution limit of the imaging, if it is certain that the measurement signal comes only from the region of this one zero position. Besides the size of the zero position, the accuracy achievable in the location determination essentially depends only on the density of the pixels of the sensor array, which is conventionally a CCD or CMOS camera, as well as the signal-to-noise ratio achieved and the width of the point spread function of the imaging. Specifically, the accuracy achievable for the location determination is even much finer than the spacing of the pixels of the sensor array divided by the imaging scale; with a good signal-to-noise ratio even much less than one nanometer, which is known to the person skilled in the art.
It is also known to use this phenomenon for the localization of individual fluorescent molecules in a specimen. A prerequisite for this, however, is that the individual fluorescent molecules lie at a distance from their respective closest neighboring molecules which is greater than the spatial resolution limit of the imaging of the specimen onto the sensor array, since otherwise the optical measurement signals received by the sensor array from the individual fluorescent molecules merge together. When this happens, the positions of the individual molecules can no longer readily be determined.
In the method known from US 2004/0212799 A1 and US 2006/0038993 A1 and all previous methods in which a structure of interest in a specimen is marked with a substance emitting a measurement signal, i.e. in particular a fluorescent substance, the density of the molecules of the substance in the specimen is regularly so large that the distance of the individual molecules from their closest neighbors corresponds only to a small fraction of the spatial resolution limit of the specimen's imaging onto the sensor array.
WO 2006/127692 A2 has disclosed a method for the high spatial resolution imaging of a structure of interest in a specimen, in which the structure of interest is marked with switchable fluorescent dyes in the form of so-called phototransportable optical markings. A subgroup of the markings is respectively activated into a state in which they can be excited to emit fluorescent light. The respective subgroup comprises so few of the markings that they lie at a distance from one another which is greater than the spatial resolution limit for imaging the specimen onto the sensor array. This makes it possible, after exciting the markings of the subgroup into fluorescence, to localize the origin positions of the fluorescent light with a resolution better than the diffraction limit which applies for the spatial resolution for imaging the specimen onto the sensor array, so that a point of the marked structure of interest is also respectively recorded with this increased resolution. The phototransformable optical markings are defined in WO 2006/127692 in that they can be switched on by an activating signal into a state in which they can be excited to emit fluorescent light. This activating signal may be the same as the excitation light which subsequently excites the markings into fluorescence. More specific embodiments of phototransformable optical markings, which are disclosed in WO 2006/127692, comprise exclusively a photoactivatable fluorescent proteins, i.e. molecules which become a fluorophore only after they have absorbed at least one light quantum, or in other words they initially need to be switched on before they are fluorescent. The activating or switching process entails a modification of the molecular structure of the molecules (relocation of atom groups or even breaking or forming a bond). The method known from WO 2006/127692 is also referred to as PALM (Photoactivated Localization Microscopy).
A similar method known as STORM (Stochastic Optical Reconstruction Microscopy) and described by Rust et al. in Nature Methods, 3, 793-796 (2006) likewise uses molecules switchable into a fluorescent state, i.e. switchable fluorescent dyes, although these are not proteins but photoswitchable organic fluorophores, specifically the fluorescent dyes Cy3 and Cy5. It is known of these cyanine dyes that they can be switched between different conformational states, more specifically isomeric states.
A disadvantage of the PLM and Storm methods is that it is not possible in them to predict when the structure of interest in the specimen will be recorded so fully that determining the position of further molecules provides no additional useful information and the method may therefore be terminated.
The range of switchable proteins and fluorophores, which may be used for the RESOLFT, PALM and STORM methods explained above, is very small compared with the total number of fundamentally known and available fluorescent dyes. Dyes which are both switchable and (in one of the switching states) capable of fluorescence, are very rare. They are therefore synthesized and optimized by elaborate methods. Added to this, the switching behavior and the fluorescent behavior depend very strongly on the chemical environment of the molecule. This applies both for switchable fluorescent proteins and for switchable organic fluorophores. This deficiency is to be regarded as fundamental, and it is associated inter alia with the fact that fluorescence and switching of the molecule are mutually competitive molecular processes which often compete with one another from the same excited state. The brightness of the switchable fluorescent dyes in their fluorescent state, i.e. the relative yield of fluorescent light from a molecule during repeated excitation, is also often only small compared with a multiplicity of nonswitchable organic fluorophores and nonswitchable fluorescent proteins. The strong restrictions due to switchable proteins or fluorophores, however, have to date being tolerated in order to obtain the high spatial resolutions achievable by the aforementioned methods for imaging structures of interest.
In so-called GSD (Ground State Depletion) microscopy (S. Bretschneider et al.: Breaking the diffraction barrier in fluorescence microscopy by optical shelving, Phys. Rev. Lett. 98, 218103 (2007)), the diffraction limit for imaging a structure marked by a fluorescent dye in a specimen is overcome by converting the respective fluorescent dye outside the respective measurement point from its electronic ground state, from which it can be excited into fluorescence by excitation light, into a dark electronic state in which it is not capable of fluorescence. This is done before exciting the remaining molecules at the measurement point into fluorescence by depopulating light with the same wavelength as the excitation light. The dark electronic state is typically a triplet state, while the ground state of the fluorescent dye is a singlet state. The molecules typically return thermally, i.e. not (optically) switched, from this dark state into the electronic ground state, so that only light of a single wavelength i.e. the excitation light is necessary for carrying out the experiment.